Receptor-effector coupling by G proteins: implications for normal and abnormal signal transduction.

0163-769X/92/1303-0536$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society

Vol. 13, No. 3 Printed in U.S.A.

Receptor-Effector Coupling by G Proteins: Implications for Normal and Abnormal Signal Transduction ALLEN M. SPIEGEL, ANDREW SHENKER, AND LEE S. WEINSTEIN Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

I. Introduction II. G Protein Structure and Function A. General features B. a-Subunit structure C. /?- and 7-Subunit structure III. G Protein Diversity IV. Subcellular Localization V. Receptor Activation of G Proteins VI. Specificity of Receptor-G Protein Coupling A. Determination of coupling preferences 1. Reconstitution of purified receptors and G proteins 2. Transfection B. Determination of coupling in situ 1. Selective a-subunit C-terminal peptide antibodies 2. Solubilization of native receptor-G protein complexes 3. Agonist-dependent radiolabeling of G proteins 4. Antisense oligonucleotides C. Comparison of experimental approaches VII. Atypical Stimulation and Inhibition of G Proteins VIII. G Protein-Effector Coupling IX. G Protein Alterations in Disease States A. Posttranslational modifications of G proteins by bacterial toxins B. Mutations of a-subunit genes 1. G protein mutations resulting in constitutive activation 2. G protein mutations resulting in functional deficiency C. Altered G protein expression or function in pathophysiology 1. Endocrine disorders 2. Tumor metastasis 3. Cardiovascular disorders 4. Neuropsychiatric disorders 5. Immunodeficiency X. Conclusions and Future Prospects

was first covered in Endocrine Reviews in 1981, when three distinct G proteins involved in visual transduction and regulation of cAMP formation were recognized (1). This topic was updated in Endocrine Reviews in 1989 by which time the number of mammalian G proteins identified had doubled, and their roles in regulation of various phospholipases and ion channels had been recognized (2). Barely 3 years later, the number of distinct mammalian G proteins recognized has again doubled, multiple G proteins have been identified in invertebrates, plants, and single-celled eukaryotes, and the number of cellular functions shown to be regulated by G proteins has increased correspondingly. Numerous excellent reviews covering various aspects of this topic have recently appeared (3-9), but given the rapidity of progress in this field, an update in Endocrine Reviews appears timely. This review summarizes recent progress in our understanding of G protein structure, function, and diversity. Particular emphasis is given to the problem of defining specificity in coupling of receptors to various G proteins, and the involvement of abnormal G proteins in human disease. Two related areas, that of G protein-coupled receptors (10) and of low molecular weight GTP-binding proteins such as the ras p21 protooncogenes (7, 11, 12), have also undergone an explosive increase in information. Detailed consideration of the structure, function, and diversity of both of these important classes of protein is beyond the scope of this review, but selected areas will be covered as they relate to the heterotrimeric G proteins themselves. Our citation of the extensive literature on G proteins is not comprehensive; instead, we have emphasized more recent references, and in some instances directed the reader to recent reviews for more detailed coverage of a particular area.

I. Introduction II. G Protein Structure and Function

'HE ROLE of heterotrimeric guanine nucleotide binding proteins (G proteins) in signal transduction

A. General features

G proteins are a subfamily within the large superfamily of GTP-binding proteins that includes ras and ras-like

Address requests for reprints to: Allen M. Spiegel, M.D., NIH, Building 10, Room 9N-222, Bethesda, Maryland 20892.

536

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 November 2015. at 23:13 For personal use only. No other uses without permission. . All rights reserved.

August, 1992

RECEPTOR-EFFECTOR COUPLING BY G PROTEINS

proteins, as well as elongation and initiation factors of ribosomal protein synthesis (4, 7). Although there is substantial diversity within the G protein subfamily, members have certain features of structure and function in common. By definition, all are heterotrimers consisting of a-, /?-, and 7-subunits, each the product of distinct genes. It is the a-subunit which shows structural and functional homology with other members of the GTPbinding protein superfamily. It binds guanine nucleotides with high affinity and specificity and possesses intrinsic GTPase activity. @- and 7-subunits are tightly but noncovalently associated to form a functional /^-complex. G proteins function as transducers of information across the cell membrane by coupling diverse receptors to effectors. They act as molecular switches with an "on-" and "off-" state governed by a GTPase cycle (Fig. 1). Receptors activated by appropriate extracellular signals (hormones, neurotransmitters, odorants, and photons of light) function as exchange factors, enabling

FIG. 1. The G protein GTPase cycle. In their basal, inactive state, asubunits contain tightly bound GDP and are associated as a heterotrimer with the 187-complex. Interaction with the intracellular portion of an agonist-bound, activated, receptor (shown in the schematic with seven putative transmembrane-spanning domains) leads to release of bound GDP and binding of ambient GTP. Binding of GTP leads to dissociation of G protein from receptor, and of a-subunit from (iy. GTP-bound a interacts with and regulates effector. The dashed line indicates that in some cases (see text) the 187-complex may also regulate effector activity. Intrinsic GTPase activity of the a-subunit leads to hydrolysis of bound GTP to GDP with liberation of inorganic phosphate. This causes dissociation of a-subunit from effector and reassociation with #7. Bacterial toxins covalently modify a-subunits, and thereby alter signal transduction. PTX blocks signal transduction by several G proteins by uncoupling them from receptors. CTX constitutively activates its substrate a8, causing agonist-independent cAMP formation. Mutations identified in a8 block signal transduction by uncoupling it from receptors (unc) or preventing GTP activation (H21a). Certain a-subunit mutations (see text) can also lead to constitutive activation of the a-subunit and effector pathway by inhibiting GTPase activity.

537

tightly bound GDP to be released and permitting GTP to be bound. Certain bacterial toxins modify G protein function by transferring ADP-ribose from NAD to specific amino acids within the a-subunit. Pertussis toxin (PTX) covalently modifies a subset of a-subunits (Table 1) and inhibits guanine nucleotide exchange by blocking receptor-G protein coupling. Since intracellular GTP concentrations exceed those of GDP by at least 1 order of magnitude, it is generally assumed that even with equal affinity for GDP and GTP, the "empty" site will be filled by GTP after GDP release. Some have suggested that a nucleoside diphosphokinase (NDPK) may be responsible for maintaining high GTP concentrations in proximity to G proteins (13). NDPK was shown to activate the small GTP-binding protein (ADP-ribosylation factor), putatively by phosphorylating bound GDP to GTP (14). No definitive evidence, however, demonstrates a role for NDPK in directly converting a-subunit-bound GDP into GTP (15). Different G proteins have been shown to possess different intrinsic rates of guanine nucleotide exchange in the absence of receptor (16-18). Binding of GTP leads to a change in a-subunit conformation (5) that causes G protein activation, and, at least in solution, dissociation of the GTP-bound a-subunit from the /3Y-complex. Recent evidence (19) suggests that G protein subunit dissociation also occurs in situ (i.e. for membrane-bound G proteins), and is indeed essential for normal effector regulation. The a subunit has been shown to play the critical, if not only, role for regulation of several effectors by their corresponding G protein. There is, nonetheless, evidence that the fiycomplex could play an important role in regulation of certain effectors including adenylyl cyclase (20-22), cardiac atrial potassium channels (23), and phospholipase A2 (PLA2) (24, 25), and in the yeast mating factor pathway (7, 9). G protein activation is terminated by a GTPase activity intrinsic to the a-subunit. Although relatively slow (-2-4 GTP hydrolyzed per min), a-subunit GTPase activity is orders of magnitude faster than that determined for members of the low molecular weight GTPbinding protein subfamily (5, 7). For the latter, distinct GTPase-activating proteins (GAPs) have been identified; no corresponding proteins have been identified for a-subunits, and this function may be subserved by a domain within the a-subunit itself (5). Binding of synthetic, nonhydrolyzable analogs of GTP leads to persistent G protein activation. Cholera toxin (CTX)-catalyzed modification of as, the G protein mediating stimulation of cAMP formation, inhibits GTPase activity and leads to persistent activation by the natural ligand, GTP. Fluoride ions (in the form of an aluminum fluoride complex) also cause persistent G protein activation. The complex binds adjacent to GDP and apparently mimics


Vol. 13, No. 3

SPIEGEL ET AL.

538 TABLE 1. Mammalian a-subunit diversity a-Subunit GB Goif

Gtl

Toxin substrate CTX CTX

Gn Gi2 Gi3 Go Gz

PTX/CTX PTX/CTX PTX PTX PTX PTX -

Gq Gu

_ -

Gt2

Expression

f Adenylyl cyclase, Ca2+ channel f Adenylyl cyclase

Rod photoreceptors Cone photoreceptors Neural > other tissues Ubiquitous Other tissues > neural Neural, endocrine Neural, platelets

| cGMP-phosphodiesterase | cGMP-phosphodiesterase

Gi5/i6

-

Ubiquitous Ubiquitous Liver, lung, kidney Blood cells

G12 G13

_ -

Ubiquitous Ubiquitous

GM

Effector

Ubiquitous Olfactory

the Y-phosphate group of GTP (26). Interestingly, despite high conservation of amino acids involved in guanine nucleotide binding across the superfamily, only asubunits are apparently capable of being activated by fluoride (26, 27). Upon hydrolysis of GTP to GDP, the a-subunit again switches conformation and regains high affinity for the ^-complex. Formation of the heterotrimer is required for high affinity coupling of G protein to receptor (28, 29). B. a-Subunit structure a-Subunits interact with guanine nucleotides, the f5y~ complex, receptors, effectors, and cell membranes, and perhaps other as yet unidentified structures. Given the multiplicity of interactions, it is not surprising that the overall amino acid sequence of a particular G protein asubtype tends to be conserved. Certain regions of all asubunits are very highly conserved (>90%), whereas others are much more distinctive for a particular subtype (. I J

| Phospholipase C-/3

? ?

a-Subunits range between 39 and 52 kilodalton (kDa) in apparent molecular weight based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five discontinuous regions of the a-subunit primary sequence (Fig. 2) represent sequence motifs highly conserved not only across the a-subunit subfamily, but also across the GTPbinding protein superfamily. Extrapolations from the ras p21 crystal structure support the idea that these asubunit regions are involved in guanine nucleotide binding. Various designations have been used for such regions (6, 7); we will refer to them as G1-G5 (5, 8). Gl has the consensus (single letter amino acid code where X is any residue), GXXXXGK(S/T), known to be involved in phosphate binding in all types of nucleotide binding proteins. The last residue in the Gl motif is presumed to participate in magnesium ion binding (35). Many asubunits have the sequence GAGESGKS for Gl, but some more recently cloned such as az (GTSNSGKS) and aq (GTGESGKS) vary from this consensus, and this may underlie their much slower rate of GDP release (17, 36, 37). Mutations of this region in a-subunits can slow GTPase activity (8, 38-40) but do not lead to powerful constitutive activation as they do in ras p21. G2 includes a highly conserved threonine residue that may interact with the magnesium ion bound to the /?and 7-phosphates of the guanine nucleotide (5). G3 has the sequence DVGGQ in most a-subunits and may be involved in the critical conformational switch engendered by replacement of GDP with GTP, and in the hydrolysis of bound GTP. The glutamine (Q) corresponds to position 61 in ras p21 which when mutated reduces GTPase activity and causes constitutive activation and malignant transformation of fibroblasts. Similar mutations in various a-subunits likewise cause reduced


August, 1992


539

G2

FIG. 2. Schematic view of the a-subunit of Gs. The figure is designed to highlight the proposed functions and general spatial relationships of different regions of an a-subunit and is not intended to represent the actual three-dimensional structure of the protein. The a8-subunit is viewed from within the cell, with the plasma membrane in the background. Bound GTP is represented as PPP-G. The subunit's amino terminus (involved in 187 binding and attenuation of GDP dissociation) and carboxyl tail (involved in receptor coupling) are depicted in proximity to the membrane, and to each other. The /37-complex is required for activation of the a-subunit by receptor, which promotes release of bound GDP. The five conserved sequences involved in guanine nuclotide binding and hydrolysis are shown as dark bands (Gl - G5). Gl is involved in binding the aand /3- phosphates of the nucleotide. The Arg201 residue in G2 is the site of ADP-ribosylation by CTX. Covalent modification or gsp mutation of this residue impairs hydrolysis of the 7-phosphate of GTP. G3 is critically involved in the conformational change that results from replacement of GDP with GTP ("switch"), and in hydrolysis of the 7-phosphate ("timer"). Mutations of discrete residues in G3 have been shown either to inhibit activation (Gly225-»Thr, Gly226-»Ala) or to markedly inhibit GTPase activity (gsp mutation of Gin227). G4 and G5 are involved in binding the guanine ring of the nucleotide. The function of the large region between Gl and G2 is unknown, but it has been proposed to act as an endogenous activator of GTPase activity ("GAP-like" domain). Three striped "effector" domains on the opposite face of the protein (residues 236240, 277-285, and 349-356) have recently been shown to be involved in the stimulation of adenylyl cyclase. The hatched region just beyond G5 (residues 367-376) is proposed to play a role in membrane attachment of a8. Modifications within the extreme C-terminal domain (unc mutant Arg389—»Pro in a8) ADP-ribosylation by PTX in several other a-subunits) produce functional uncoupling of receptor and G protein. Numbering of residues refers to the 394 amino acid form of a8. Additional details and references are found in the text.


540

SPIEGEL ET AL.

GTPase activity and constitutive activation of effector pathways (38, 39, 41-43). In as, mutation of either of the adjacent G3 glycine residues (DVGGQ) blocks activation by GTP (44, 45). This contrasts with ras p21, in which mutation of position 59 glycine or position 61 glutamine causes equivalent constitutive activation. Mutation of a glycine (DVGGQ) in ai2 also blocks GTP activation (46). The G4 region has the sequence NKXD and is involved in binding to the purine ring; this region confers specificity for guanine as opposed to other nucleotides. G5, usually TCAXDT in a-subunits, may also interact indirectly with the guanine nucleotide. CTX catalyzes ADP-ribosylation of an arginine residue (Arg201 in the long form of as) within the G2 region. Ordinarily only as is a good substrate for CTX despite the fact that this arginine residue is conserved in all other a-subunits. Whether this reflects unique conformational requirements conferred by neighboring or distant amino acid residues is unclear. Under certain conditions (receptor activation and removal of guanine nucleotide), other a-subunits also become CTX substrates (47). CTX-catalyzed ADP-ribosylation of such a-subunits may require creation of an "empty site" (48). CTX modification, not only of as, but also of other substrates such as ai2, inhibits GTPase activity and causes constitutive activation (49). Mutation of the same arginine residue inhibits GTPase activity and causes constitutive activation comparable to that caused by covalent modification of the residue by CTX (46, 50, 51). This may reflect proximity of this residue to the 7-phosphate of bound GTP (Fig. 2). PTX also catalyzes ADP-ribosylation, but the acceptor is a cysteine residue fourth from the carboxy terminus (52, 53). This leads to uncoupling of the G protein from the receptor but does not modify other key aspects of G protein function such as guanine nucleotide exchange and GTPase activity (54). Mutation of an arginine residue, sixth from the carboxy terminus of as also leads to uncoupling from receptor (55, 56). This and other evidence suggest that portions of the carboxy terminus are critical in receptor interactions. The highly variable region between Gl and G2 had originally been speculated to be involved in effector interactions (30), but more recent data from chimeric G proteins (57-59) and sitedirected mutagenesis (34, 60) place the effector interaction region within the carboxy-terminal third of the asubunit. The region between Gl and G2 may represent an endogenous GAP for a-subunits (5). Extrapolations from the ras p21 crystal structure suggest that the amino- and carboxy termini of a-subunits may be in close proximity and are oriented toward the membrane surface (32). There is evidence from chimeric constructs that both termini may be involved in suppressing GDP dissociation and in interaction with the

Vol. 13, No. 3

/?7-complex (8,59,61). Proteolytic cleavage of the aminoterminal 1-2 kDa portion of a-subunits eliminates interaction with the /fy-complex (62, 63). For certain asubunits, myristoylation of an amino-terminal glycine residue is required for high affinity interaction with the 07-complex (64, 65). Interestingly, despite the evidence for involvement of the carboxy terminus in receptor coupling, mastoparan (a G protein-coupled receptor mimetic peptide, see below) was shown to cross-link to a cysteine residue near the amino terminus of the a-subunit (66). This may reflect proximity of the amino- and carboxy termini of a-subunits, and the interaction of both receptors and the /37-complex with these regions. C. /?- and y-Subunit structure The /37-complex: 1) suppresses GDP dissociation, thereby maintaining a-subunits in their inactive state, 2) is required for high affinity coupling of G protein to receptor, 3) may be involved in direct regulation of certain effectors, and 4) binds to cell membranes. (3- and 7-subunits tightly associate with each other, and the (3ycomplex clearly interacts with a-subunits. Whether or not the /?7-complex directly interacts with receptors is not yet clear. The /ity-complex associates with a 33 kDa protein kinase substrate in retinal photoreceptors (67), and jS-immunoreactivity coelutes with the 33 kDa protein in a 70 kDa fraction on gel permeation chromatography of pineal cytosol (68). The physiological significance of the presumed association between the 33 kDa protein and the /^-complex has not been defined. A variety of studies have provided some insights into the regions of j8- and 7-subunits involved in particular functions, but in comparison with a-subunits, the information is sketchy. Mammalian jS-subunits are generally 340 amino acids long and migrate as 35-36 kDa proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They are composed of eight repetitive segments of approximately 40 amino acids each that contain characteristic motifs, in particular a tryptophan-aspartic acid pair (6). This motif, termed "WD-40," has been found in a variety of apparently functionally unrelated proteins, and as yet its functional significance is unclear (6). 7-Subunits are roughly 70 amino acids long and vary in apparent size from 5-10 kDa on gel electrophoresis depending on buffer conditions. They contain a conserved carboxy-terminal tetrapeptide (cysteine, aliphatic, aliphatic, L/S) that is the site of posttranslational processing critical for membrane binding of the (87-complex (69) (see Section IV). This region may be involved in a-subunit and possibly receptor interaction (8, 70, 71). The native /fy-complex is quite resistant to tryptic cleavage. Although there are multiple potential cleavage


August, 1992


sites, a single residue (Arg129) in the /?-polypeptide is the only site actually cleaved. This results in an aminoterminal approximately 14 kDa fragment and a 26 kDa carboxy-terminal fragment (62, 72), both of which remain associated with the 7-polypeptide under native conditions (73). Cross-linking studies indicate that the a-subunit interacts with the 26 kDa /3-subunit tryptic fragment (74).

III. G Protein Diversity G proteins were first identified and characterized by functional criteria. G8 and G; represented G proteins mediating stimulation and inhibition, respectively, of adenylyl cyclase; Gt (transducin) represented the photoreceptor G protein involved in coupling light-activated rhodopsin to cGMP-phosphodiesterase. Susceptibility to covalent (and functional) modification by bacterial toxins also proved useful in identifying, and to some extent discriminating between, G protein a-subunits. The first such effect of PTX to be recognized was prevention of receptor-mediated adenylyl cyclase inhibition (53). Hence "Gi" was the first recognized PTX substrate. It soon became clear that PTX could block receptor regulation of other effector pathways including certain potassium and calcium channels. This led to confusion as to the identity of the PTX-sensitive G proteins involved in regulating such pathways. Purification and biochemical characterization of G proteins revealed unanticipated diversity in the subfamily. Proteins corresponding to Gs, Gt, and "Gi" were purified (3, 28, 75-78), but so were novel G proteins of unknown function. For example, the most abundant G protein in brain turned out to be a novel PTX substrate, termed Go (79, 80). Further purification efforts revealed additional biochemical heterogeneity compatible with multiple Gi and Go subtypes (81, 82). Immunochemical evidence also suggested a multiplicity of G; subtypes (83). Molecular cloning of complementary DNAs (cDNAs) encoding putative a-subunits provided clear evidence for multiple a; subtypes (84). Polymerase chain reaction-based cloning (85-88) has now brought the number of distinct genes encoding mammalian a-subunits to at least 15 (Table 1). Further diversity is achieved by alternative splicing of the as (four forms) and a0 (two forms) genes (89-91). For a given asubunit subtype, the amino acid sequence is highly conserved (generally >95%) across mammalian species. Mammalian a-subunits can be grouped into four classes (6) based on amino acid identity and presumed evolutionary distance: 1) a8 and aoif, 2) a t i, at2, a-n, ai2, ai3, a0, and az, 3) aq, an, au, ai5/i6, 4) ai 2 , a i3 . a1B is apparently the murine equivalent of human ai6 (6), bovine liver asubunit cDNAs termed «LI and aL2 (92) correspond to

541

ai4 and an (6), respectively, and a human retinal cDNA termed ay corresponds to an (93). Another novel cDNA, «gust, has recently been isolated from rat taste buds (94). Northern analysis and immunoblotting have helped define the range of expression of the encoded proteins (Table 1). This ranges from highly restricted, as for the G proteins involved in visual and odorant transduction, to ubiquitous for G proteins (Gs and Gi2) involved in regulation of the ubiquitous second messenger, cAMP. The arbitrary nomenclature (letters and/or numbers used to designate the subtypes) obviously no longer reflects function, since in many cases definitive knowledge concerning the function(s) of a given subtype is lacking. Table 1 gives some indication of the effectors regulated by various G proteins, and this, along with specificity of receptor interactions, is discussed in later sections. There is also diversity of mammalian /3- and 7-subunits (6). There are at least four distinct /3-subunit cDNAs, and at least as many 7-subunits (95). How these actually combine to form distinct (3y -complexes is not yet clear. There is some indication that different (3ycomplexes may have distinctive properties with respect to a-subunit (96) and receptor (97-99) interactions, but much more work is needed to clarify this. G proteins represent a highly conserved signal transduction mechanism. a-Subunits have been identified (most often by cDNA cloning) in nonmammalian vertebrates (100), in invertebrate animals such as fruit fly [see Refs. 85, 101, and 102 and references therein for examples], squid (103), and roundworm (104), in slime molds (105) and yeast (7, 9), and in plants (106-108). /3And 7-subunits have also been identified in such organisms, for example in yeast (109), fruit fly (110), and squid (111). Given the evolutionary distances, there is often surprisingly high primary sequence conservation between the mammalian proteins and their nonmammalian counterparts (6, 7). For some G protein subtypes, not only structure, but also function, appear to be conserved. G8, for example, stimulates cAMP formation in fruit flies (112) as in mammals. In other instances, G proteins have evolved for unique functions such as the mating factor pathway in brewer's yeast (9) and cellular aggregation in slime molds (105). As with the mammalian G proteins, molecular cloning has revealed novel G proteins (for example Ref. 101) for which specific functions (and relevant receptors and effectors) remain to be identified.

IV. Subcellular Localization None of the G protein subunits possesses hydrophobic, putative membrane-spanning domains. Nonetheless, after cell lysis and fractionation, G proteins are typically found in the particulate fraction and require detergent


542

SPIEGEL ET AL.

to release them from the membrane. This has generally been assumed to reflect tight association with the cytoplasmic surface of the plasma membrane. The molecular basis for this membrane association has recently been reviewed in some detail (69). Only a brief summary, and some more recent data will be given here. Despite earlier evidence obtained with phospholipid vesicles (113), asubunits and /fy-subunits appear to be capable of associating with the plasma membrane independent of each other. Certain a-subunits such as a ; and a0 require amino-terminal, cotranslational modification with myristic acid in order to become membrane-bound. The yeast mating factor pathway a-subunit, GPA1, also undergoes myristoylation, but this is not obligatory for its membrane association (114). For all of these a-subunits, however, myristoylation appears to be essential for highaffinity interaction with the /37-complex (64, 65). Failure to undergo myristoylation prevents GPA1 from inhibiting mating factor signaling, presumptively because it cannot associate with STE4 and STE18 gene products (the mating factor pathway jfry-complex) (114). Other asubunits such as as, and perhaps some of the more recently identified ones (aq, an), do not undergo myristoylation and yet are membrane-bound (69). The basis for this membrane association is not clear. A region near the carboxy terminus (residues 367-376 of the long splice variant) of as has been implicated in membrane association (115). Interestingly, aq and an share a common sequence with as within this region (TENIR in single letter code), but further studies are needed to clarify the role of this or other regions in membrane association of nonmyristoylated a-subunits. a u is unique in that it is associated with the cytoplasmic face of a highly specialized membrane, that of the outer segment disc in rod photoreceptors. The purified protein lacked detectable myristate (116), although the sequence is compatible with the myristoylation consensus (69). This may explain the ease with which the protein is released from rod outer segment membranes in buffers lacking detergent. G protein 7-subunits undergo three sequential posttranslational modifications of their carboxy terminus that are critical for membrane binding of the 07-complex (69). Isoprenylation, the first modification of the 7subunit carboxy terminus, may involve addition of a farnesyl or geranylgeranyl moiety depending on the last amino acid of the 7-subunit (69). For 7 t , carboxy-terminal processing has been shown to be required for high affinity interaction with the a-subunit and receptor (rhodopsin) (71). Biochemical studies performed to date do not answer several important questions regarding the actual state of G protein subunits associated with the plasma membrane. Are they part of macromolecular assemblies? If so, what other proteins if any participate? Do they leave

Vol. 13, No. 3

the membrane to interact with proteins in other subcellular compartments under physiological or even pathophysiological conditions? Some evidence suggests that a-subunits may exist as large oligomers (117) and that they may interact with cytoskeletal elements such as tubulin (118). Data suggesting that a-subunits may, in fact, be translocated from the membrane upon agonist stimulation (119, 120) and that this process may play a role in signal transduction (121, 122) have been presented. Studies with newer cell biological techniques will help define these issues more definitively. There is evidence that G proteins may occur in the cell in specialized regions of the plasma membrane or in compartments other than the inner surface of the plasma membrane. In polarized epithelia, there may be differential localization of a; subtypes to apical and basolateral membranes (123). a0 appears to be preferentially localized to nerve endings where it may associate with, and be regulated by, the GAP-43 nerve growth cone protein (124). Low molecular weight GTP-binding proteins are known to be involved in vesicle transport of proteins throughout the cell (4), but recently, ai3 has been localized to Golgi membranes, and it, or a closely related G protein, may be involved in protein trafficking through Golgi vesicles (125-127).

V. Receptor Activation of G Proteins Identification and characterization of members of the family of seven-transmembrane-segment (7-TMS) receptors is progressing rapidly (10, 128-130). When an agonist binds to one of these receptors, the molecular perturbation is relayed from the membrane-embedded helices to the cytoplasmic face of the protein. Analysis of the effects of deletion mutants, chimeric receptors, effects of peptides corresponding to portions of receptor sequence, and antibodies directed against different cytoplasmic regions have been used to map potential sites of interaction between receptor and G protein (10, 128, 129, 131-139). It has become evident that the concerted participation of several intracellular receptor domains is involved in coupling to G proteins. These domains include the second cytoplasmic loop, the third cytoplasmic loop, especially its N- and C-terminal segments, and the N-terminal segment of the cytoplasmic tail. Portions of a receptor's intracellular loops are important both in determining relative affinity for different G proteins, and in transmitting the signal produced by agonist binding. A mutant /32-adrenergic receptor (AR) with a seven amino acid deletion (residues 267-273) in the carboxyterminal region of its third cytoplasmic loop retains its ability to bind Gs without being able to transmit a stimulatory signal (132). Although this finding suggests that binding and activating functions may be subserved by


August, 1992


discrete cytoplasmic domains, relatively small peptides are capable of acting as receptor-mimetics (140-143). For example, a peptide corresponding to residues 259-273 of the J02AR is able to potently mimic agonist-occupied receptor by binding and activating G8 (135). Furthermore, the peptide retains the native receptor's relative selectivity for G8 us. Gi. Protein kinase A-catalyzed phosphorylation of a serine residue that corresponds to a site that can be modified during receptor desensitization not only blunts the ability of the peptide to activate Gs but also potentiates its otherwise weak activation of Gi. These findings emphasize the importance of the C-terminal domain of the third intracellular receptor loop in activating G proteins and raise the intriguing possibility that coupling specificity is not a static property of receptors, but rather one that might be dynamically regulated in the cell. Substitution of amino acid residues in the corresponding C-terminal segment of the third intracellular loop of the «IBAR produces marked agonist-independent stimulation of cellular phosphatidylinositol turnover, as if the unoccupied mutant receptor is stabilized in the active conformation normally assumed by the agonist-bound wild type receptor (133,139). Furthermore, these mutant receptors are capable of inducing neoplastic transformation when overexpressed in cells (144). Mutations in critical GTPase domains of G protein a-subunits that result in a prolonged lifetime for the stimulatory conformation have been described in several human tumors. Could spontaneous occurrence of activating mutations in the C terminus of the third intracellular loop of certain 7-TMS receptors play a similar role in human disease by effectively eliminating agonist dependence? Although there are examples of diseases due to mutations in the tyrosine kinase class and other classes of cellular receptors, the only well-characterized 7-TMS receptor disease is retinitis pigmentosa. More than 30 different mutations of rhodopsin have been described in the disease, which is characterized by retinal degeneration and progressive blindness (137,145,146). The catastrophic effect of some of these rhodopsin mutations may be secondary to an accumulation of abnormally folded or targeted protein in the photoreceptor cells. It can be imagined that mutations that affect binding, activation, or desensitization of other 7-TMS receptors will be discovered in some human diseases, including syndromes of hormone-resistance [e.g. congenital nephrogenic diabetes insipidus (147), pseudohypoparathyroidism type Ib (148)] and diseases involving unregulated cell function or growth [e.g. cancer, atherosclerosis (144)].

VI. Specificity of Receptor-G Protein Coupling A living cell contains a variety of homologous 7-TMS receptors, G proteins, and effector proteins. Delineating

543

the actual degree of specificity of receptor coupling to cellular effectors is needed in order to help understand complex cellular signaling. It has become evident that receptor-effector coupling by G proteins is neither absolutely specific nor totally promiscuous. First, multiple receptors in a cell may converge on a single G protein subtype. A single, molecular subtype of receptor has explicitly been shown to be capable of coupling to more than one type of G protein in artificial reconstitution (29,149,150) and transfection studies (41,151-153), but whether this occurs naturally has been harder to prove because of the possibility of receptor heterogeneity in vivo (154,155). Selective immunoprecipitation of a single molecular type of muscarinic receptor from three different tissues, using a defined monoclonal antibody, recently demonstrated that this receptor appears to be associated with a different complement of PTX-sensitive G proteins in each area (156). A variety of different approaches, each with its own advantages and disadvantages, have been used to define the specificity of coupling between receptors and effectors provided by G proteins (Table 2). A. Determination of coupling preferences 1. Reconstitution of purified receptors and G proteins. The first reconstitution experiments used purified or only partially purified components (29). Conclusions about the specificity of coupling are necessarily limited by the homogeneity of the receptor and G protein components used in the reconstitution. Receptor purified from tissue may contain a mixture of molecular subtypes. Even when great care is taken to chromatographically resolve and purify a-subunits from mammalian tissue, the possibility remains that an unknown, comigrating species may contribute to the activity of a supposedly "pure" fraction. Although reconstitution experiments continue to be performed with purified native proteins (157-159), investigators have begun using recombinant proteins purified TABLE 2. Methods used to define the specificity of receptor-G protein coupling Determination of Coupling Preferences Reconstitution of purified proteins Transfection of receptor cDNA into cells differing in endogenous G proteins Cotransfection of receptor and a-subunit cDNA Determination of Coupling in Situ PTX sensitivity Selective a-subunit C-terminal antibodies Solubilization of native receptor-G protein complexes followed by immunoblotting Agonist-dependent labeling of G proteins with [32P]GTP azidoanilide or [32P]ADP-ribose plus CTX Selective a-subunit antisense oligonucleotides


544

SPIEGEL ET AL.

from cells such as Escherichia coli (16, 99, 160, 161) or Sf9 (149, 162) that can be made to overexpress only a single receptor or a-subunit subtype. One drawback of using E. co/i-derived a-subunits is that they are functionally less active and have lower affinity for fiy- than purified mammalian a-subunits, presumably because of inadequate posttranslational processing (65). Investigators have therefore recently turned to the Sf9 baculovirus expression system as a potential source of abundant, appropriately myristoylated a-r and ao-subunits (162). Even reconstitution experiments with recombinant receptors and/or a-subunits have typically employed (3ysubunits purified from bovine brain, which is known to contain at least two distinct types of fi (163) and at least three types of y (95). Until the functional consequences of /?- and 7-heterogeneity are better understood, conclusions about coupling specificity between reconstituted asubunits and receptors should be interpreted cautiously (97,99,149). For example, different preparations of brain 07 have been shown to differ markedly in their ability to help reconstitute high-affinity complexes of brain yaminobutyric acid receptors and a0 (98), or turkey /3AR and the short form of as (99). The vehicle chosen for protein reconstitution is usually artificial phospholipid vesicles, but it is also possible to reconstitute some transduction systems in a detergent solution (159), or in a more complex, natural membrane environment. Investigators have used treatment with Nethylmaleimide (98, 161, 164) or PTX (16, 165, 166) to uncouple the endogenous Gi and Go proteins in membranes, and then tested the differential ability of purified a-subunits to reconstitute with native receptors. PTXtreated rat hepatocyte membranes have recently been used to show that purified a;3 is capable of restoring inhibitory coupling between native angiotensin II receptors and adenylyl cyclase (166). Membranes from E. coli that were expressing human /?AR have also been used as the vehicle for recombinant a-subunit reconstitution experiments (167). Reconstitution of a functional receptor-G protein complex can be measured with agonist-stimulated GTP7S binding and GTPase activity, or high-affinity agonist binding, but the measures chosen to define "selective" coupling should be carefully defined. For example, full concentration response curves with a-subunits were generated in order to conclude that the purified bovine pituitary D2 receptor not only has greater apparent affinity for Gi2, but also produces a greater maximal stimulation of that subtype in comparison to Giu Gi3, or Go (157). In contrast, the Ai adenosine receptor has 10-fold higher affinity for ai3, but produces the same maximal stimulation of all PTX-sensitive a-subunits (161). Just as affinity and intrinsic efficacy are independent prop-

Vol. 13, No. 3

erties that are both important in determining the pharmacological and physiological actions of a drug, the same two properties, when applied to the agonist-receptor complex, may be critical in explaining the biological specificity of receptor-G protein coupling (157). 2. Transfection. Stable transfection of receptor cDNA into cells with a particular complement of endogenous G proteins has been used to assess receptor-G protein coupling preferences (151-153,168-169). This strategy may be broadened by cotransfecting these cells with different a-subunit cDNAs (170). A transient cotransfection system has recently been described in which combinations of both receptor and a-subunit cDNA are transiently introduced into mammalian cells (41, 42, 51). Another system that may soon be exploited to answer questions about receptor-G protein coupling preferences is the Saccharomyces cerevisiae mating pathway. Taking advantage of the homology between mammalian and yeast signaling components, the cDNAs for /32AR and a8 have been coexpressed in yeast, and coupling has been demonstrated via activation of the host organism's downstream effector pathway (171). Transfection of cells with receptor cDNA has been used to determine the types of effector pathways activated by a defined receptor subtype. Many receptors are capable of stimulating adenylyl cyclase through G8. Another group of receptors {e.g. a2AR, dopamine D2, 5hydroxytryptamineiA, muscarinic m2 and m4) is linked to inhibition of adenylyl cyclase but in some cells is also capable of stimulating phospholipase C (PLC) via a PTX-sensitive G protein (168, 172-175). A third group (e.g. aiAR, muscarinic mx and m3) is primarily coupled to stimulation of PLC via a PTX-insensitive G protein (172,175). Other receptors, such as those for TSH (176), LH (177), PTH (178), and the tachykinins (179), may be capable of mediating stimulation of both adenylyl cyclase and PLC. Overexpressing the cDNA that codes for a single molecular type of receptor or a-subunit in a novel environment may circumvent the usual uncertainty about component heterogeneity, but the physiological significance of the results of these experiments may be challenged because of the potentially artificial stoichiometry and spatial relationships of the components compared to those in normal cells. Other biochemical events in situ may influence coupling specificity and allow for preferential activation of selective signaling pathways. While reconstitution and transfection approaches help reveal possible couplings, other approaches are required to identify the couplings that occur naturally in living cells. B. Determination of coupling in situ 1. Selective a-subunit C-terminal peptide antibodies. The first tool used to define specificity of coupling in native


August, 1992


membranes was PTX. Because PTX treatment renders G protein substrates incapable of receiving receptor signals, inhibition of pathways by the toxin provides evidence that one or more forms of G; and Go are involved but can not provide more specific identification. Selective C-terminal peptide antibodies bind to the same region of the a-subunit that is modified by PTX and similarly prevent activation by receptor. The utility of these antibodies in determining the specificity of receptor-G protein coupling in broken-cell systems is well documented. Manifestations of agonist-mediated signaling that can be selectively disrupted with antibodies include GTPase stimulation (180-183), adenylyl cyclase stimulation or inhibition (182, 184, 185), high-affinity agonist binding (186, 187), and PLC stimulation (188). Microinjection of C-terminal antibodies to ao and ai2 into living cells is capable of disrupting a2AR-mediated inhibition of calcium channels (154) and serum-stimulated DNA synthesis (189), respectively. High concentrations of the synthetic a-subunit C-terminal peptides that have been used to generate antibody may themselves be capable of uncoupling receptor-G protein complexes (180, 190). Relatively high concentrations of antipeptide antibody are required to disrupt receptor-G protein coupling, for even in an affinity-purified preparation only a small proportion of antibodies are expected to have high affinity for the native a-subunit conformation (188). Another limitation of the C-terminal antibodies is their inability to discriminate between pairs of a-subunits that share a common C-terminal sequence, such as aa and a;2, or an and aq (183, 184). 2. Solubilization of native receptor-G protein complexes. Under certain conditions, 7-TMS receptors may be solubilized and purified from tissue in the form of native, high affinity receptor-G protein complexes. In the case of the D2 dopamine receptor (191), Ai adenosine receptor (158,192), somatostatin receptors (187,193), C5a receptor (194), and the m2 muscarinic receptor (156, 195), selective antisera have been used to identify the particular G protein subunits that were stably associated with the receptors at the time of solubilization. This approach may be especially valuable in elucidating the natural role of /fy-heterogeneity in receptor coupling (158,193). 3. Agonist-dependent radiolabeling of G proteins. Selective agonist-dependent radiolabeling of G proteins with [32P] ADP-ribose plus CTX, or [32P] GTP azidoanilide, has recently been used to identify receptor-G protein coupling in native membranes. Although they do not normally serve as substrates for ADP-ribosylation by CTX, members of the G; family are rendered susceptible to this covalent modification when they are activated by the appropriate agonist-bound receptor (47, 152). A more

545

general method is based on the fact that an agonistoccupied receptor will promote guanine nucleotide exchange only with those a-subunits that it physically contacts. Selective incorporation of the photoreactive 32 P-labeled azidoanilide analog of GTP after incubation with agonist and exposure to UV light reflects those naturally occuring collisions (155, 196-198). This strategy was recently used to show that two different members of the aq family couple to vasopressin Vi receptors in rat liver (198). In both methods, the particular a-subunits that are covalently radiolabeled as a result of receptor activation in native plasma membranes can be subsequently identified using selective antisera. 4. Antisense oligonucleotides. The latest approach used to define which particular G protein is involved in a signaling pathway is microinjection of selective a-subunit antisense oligonucleotides into the nuclei of living cells (199). Injection of these oligonucleotides into GH3 cells selectively decreased G protein expression and elegantly demonstrated that activated muscarinic and somatostatin receptors inhibit voltage-sensitive calcium channels by specific coupling to Goi and Go2, respectively. C. Comparison of experimental approaches Among receptors, the a2AR, especially the a2A-molecular subtype, has been the most thoroughly studied regarding selective coupling to different G proteins. Different conclusions have been reached about a2AAR coupling selectivity depending on the methods used. For example, reconstitution of homogeneous a2A-receptors overexpressed and purified from COS cells with purifed recombinant a-subunits from E. coli, plus brain /fy-subunits, reveals a certain rank order (ai3 > aa > ai2 > a0 » a8) for maximal stimulation of GTPase activity (150). Overexpression of a2A-receptor cDNA in fibroblasts reveals coupling to both Gi2 and Gi3 (151-153), but only coupling to G;2 has been detected in the receptor's natural platelet membrane environment (184). Recent studies show that porcine a2A-receptors are also capable of coupling to PTX-insensitive aq- and az-subunits in a transient cotransfection assay (41, 42). Given that signaling pathways associated with stimulation of a2AR have consistently been shown to be PTX-sensitive, the physiological significance of these latest results remains to be seen. The m2 muscarinic receptor provides another example of discrepant conclusions about coupling based on the system that is studied. Reconstitution experiments reveal preferential coupling of recombinant m2 receptor to Go and Gz, compared to Gn (149), but a different, tissuespecific pattern of coupling of this receptor is revealed by solubilization of native receptor-G protein complexes from rat heart and brain: coupling to Go in atrium, G;2


546

SPIEGEL ET AL.

and Go in ventricle, and G;i, Gi2, and Go in cerebellum (156). As mentioned earlier, reconstitution and tranfection experiments may measure interactions between signaling components that are occurring at unnatural concentrations and in unusual contexts compared to native membranes. Nevertheless, because the investigator can control the concentrations of individual proteins and other assay components, reconstitution experiments remain uniquely capable of quantifying the molecular mechanisms involved in signal transduction (29). Another advantage of the reconstitution and transfection approaches is that they can be exploited to explore the structural and mechanistic basis of receptor-G proteineffector coupling by using site-directed mutagenesis of the components. Despite these advantages, investigators will continue to require techniques that identify the couplings that are physiologically relevant in a particular tissue.

VII. Atypical Stimulation and Inhibition of G Proteins The wasp venom peptide mastoparan directly promotes guanine nucleotide exchange and G protein activation (140, 141). This activity of mastoparan is related to its structure, an amphipathic helix that apparently mimics an intracellular region of a 7-TMS receptor by presenting its cationic face to the carboxy terminus of the a-subunit (141, 200). Other peptides such as substance P and the polyamine mast cell activator 48/80 may produce similar effects in a receptor-independent manner (201). Evidence is beginning to accumulate that endogenous cellular proteins other than the traditional 7-TMS receptors may operate as promoters of guanine nucleotide exchange for G proteins. For example, Gi2 apparently mediates activation of cellular calcium channels and DNA synthesis produced by the insulin-like growth factor II receptor, a single membrane-spanning protein with a cytoplasmic domain that lacks tyrosine kinase activity (142, 189, 202). A peptide corresponding to residues 2410-2423 of the cytoplasmic portion of the receptor can activate Gi2 in a manner analogous to mastoparan (142). Observations about mastoparan peptide binding (66) and insulin-like growth factor II peptide activation of G proteins (203) may provide clues about regions of the «subunit other than its C terminus that are involved in coupling to receptors. It is possible that some of the members of the growth factor receptor tyrosine kinase family also couple to G proteins. For example, although many cellular effects of epidermal growth factor (EGF) are mediated by receptor tyrosine phosphorylation, the EGF receptor has recently been shown to stimulate cardiac adenylyl cyclase via

Vol. 13, No. 3

activation of Gs (204) and also appears to couple to PTXsensitive G proteins involved in the PLC (205, 206) and PLA2 (207) signal transduction pathways. Evidence for interactions between the insulin receptor and PTX-sensitive G proteins in an artificial context (29) and in normal cell membranes (208-211) has also been presented. Both EGF and insulin promote incorporation of [ 32P]GTP azidoanilide into multiple a- subunits in pancreatic membranes (212). Multimeric antigen receptors are another type of receptor whose function has traditionally been attributed to activation of a kinase cascade. Although some studies have suggested a role for G proteins in immunological activation of T lymphocytes (213, 214), B lymphocytes (215, 216), and mast cells (217), there is still no explicit evidence for association of heterotrimeric G proteins with an antigen receptor. The physiological significance of the PTX-insensitive ai 5 and «i6 subunits recently found in lymphoid and myeloid cells remains to be defined (88). The mechanism by which bacterial endotoxin triggers activation of Gi2 in macrophages also deserves investigation (218). GAP-43 is an intracellular neuronal protein associated with the growth cone at the tip of elongating dendrites and axons. It was recently discovered that GAP-43 is a weak promoter of guanine nucleotide exchange on Go, the major noncytoskeletal component of growth cone membranes (124, 219). The mechanism of action of GAP43 differs from that of transmembrane receptors, and the intriguing possibility that G proteins can be stimulated by signals arising from within the cell certainly merits further attention. Another group of atypical G protein activators could include cell to cell adhesion receptors and the integrins, heterodimeric receptors that bind extracellular matrix proteins (220). A PTX-sensitive G protein has been implicated in the process of neurite outgrowth that is triggered by homophilic binding of the neural cell adhesion molecule (NCAM) or N-cadhedrin (221), and migration of murine melanoma cells in response to matrix proteins such as fibronectin, laminin, or collagen may involve activation of Gi2 (222, 223). The possibility has been raised that certain cellular proteins may naturally inhibit G protein function. For example, the complex of ras p21-GAP is able to uncouple muscarinic m2 receptors from the Gi protein that mediates activation of atrial potassium channels, although the exact mechanism and significance of this phenomenon are unknown (224, 225). The fact that APC and MCC, two proteins encoded by genes recently implicated in colorectal tumorigenesis, have slight similarity to a short region of the third intracellular loop of the m3 muscarinic receptor (226) has provoked speculation that the normal function of these proteins might be to bind


August, 1992


and inhibit Gq (227), a notion that awaits experimental validation. Although the ability of certain G protein subunits to serve as substrates for phosphorylation is often mentioned as a possible regulatory mechanism, little is known about the physiological significance of this phenomenon in mammalian systems. The a-subunit of Gz, but not other G proteins, has recently been shown to be phosphorylated on a serine residue by agents that activate protein kinase C in platelets (228). Insulin and EGF receptors stimulate tyrosine phosphorylation of a^, a0-, and 7-subunits in an artificial reconstitution assay (29), and «i2 has been reported to be a kinase substrate in rat hepatocytes (229) and U937 macrophages (218). VIII. G Protein-Effector Coupling The progress that has been made in understanding the .interaction of G8, Gi} and Go proteins with multiple ion channels has recently been reviewed in detail (230, 231). No attempt will be made here to provide a comprehensive review of G protein-effector coupling, but recent developments and possible directions for future research will be highlighted. Some of the same methods used to define the specificity of receptor-G protein coupling, including protein reconstitution, cDNA transfection, the use of C-terminal a-subunit antibodies, isolation of complexed components, and antisense oligonucleotide technology, are applicable to define specificity of G protein-effector coupling, and the same caveats about interpretation of results should be noted. Activation of effector pathways not normally known to be associated with a particular receptor in vivo can often be observed when receptors are overexpressed in a heterologous system, and some of these effects may be indirect, or of questionable physiological significance (175, 232-234). Conclusions about coupling specificity have been shown to depend on the level of receptor expression and the choice of cell used for transfection, including the type of G proteins and effectors the cell contains. Although reconstitution and transfection experiments suggest that several different members of the Gi family are capable of activating atrial potassium channels (235), inhibiting calcium channels (16), and mediating the inhibition of adenylyl cyclase (41, 51, 153, 166, 168), it is largely unknown which members naturally perform these functions in a particular cell. A specific role for ai3 in stimulation of sodium channels in renal epithelial cells has recently been suggested (236). Regarding the molecular mechanism of effector stimulation by G proteins, only the stimulation of retinal cyclic GMP phosphodiesterase by a t has been accessible to detailed investigation (29,237,238). Now that multiple

547

adenylyl cyclases have been cloned and characterized (21, 239-241), better understanding of that system should be expected as well. So far, the mechanism by which G; proteins inhibit adenylyl cyclase activity remains enigmatic, and the results of recent reconstitution and transfection experiments have generated apparently conflicting conclusions. High concentrations of recombinant ant ai2, and ai3 from E. coli are incapable of inhibiting purified brain adenylyl cyclase activity (16), but mutationally activated versions of all three subunits lead to decreased cAMP production when transiently expressed in fibroblasts (41, 51). In the presence of as, 07-subunits directly inhibit type I adenylyl cyclase, cause activation of the type II and IV enzymes, and have little or no effect on other forms (21, 22). A novel mechanism of adenylyl cyclase inhibition by calcium has recently been described in NCB-20 cells (242). The physiological significance of all these results remains to be explored. In some tissues the G proteins coupled to stimulation of PLC are PTX-sensitive, but in most tissues they are not. For many years the molecular identity of PTXinsensitive G proteins remained elusive. The mystery was recently solved, as several groups were independently able to purify and clone members of a novel, ubiquitously distributed family of G proteins known as Gq (36, 85, 92, 93, 243). The a-subunits of this family, including aq and an, have unequivocally been shown to stimulate the (31isozyme of PLC (37, 43, 244, 245). Homologous Gq proteins have been found in Drosophila eye (102) and turkey erythrocytes (246). Members of the Gq family bind GTP7S poorly, probably because of unusually slow GDP release in the absence of receptor (36, 37, 246). Most of the other known a-subunits, including aa, az, an, ai2, and two forms of a0, are unable to stimulate PLC-01 (43, 244, 245, 247). The effectors normally associated with Gi2, G13, and Gz remain unknown. In terms of receptor and effector coupling preferences, Gz bears a superficial resemblance to other members of the G; family (41,149), but its distinctive cellular distribution (248), slow intrinsic GTPase (17), and ability to be phosphorylated (228), suggest that it may serve a specialized function. The failure of a0 to stimulate PLC-01 (43, 247) contrasts with previous results from Xenopus oocytes suggesting that purified forms of Go function as PTXsensitive activators of the PLC pathway (249, 250). In fact, novel enzyme isoforms (251) may be involved in the case of PTX-sensitive PLC stimulation, such as that found in neutrophils and HL60 granulocytes. The possibility that some isoforms of PLC may be regulated by 187-subunits also remains to be explored. Phosphoinositide-specific PLC isozymes are not the only effector enzymes that hydrolyze membrane phospholipids. Evidence has accumulated that G proteins are involved in the activation of other such enzymes, includ-


548

SPIEGEL ET AL.

ing PLA2 (24, 25, 58, 252-254) and phospholipase D (234, 255, 256). The fiy-complex was found to stimulate production of arachidonic acid by retinal and cardiac PLA2 (24, 25), and a role for «i2 in agonist and calciumstimulated fibroblast PLA2 activity has also been proposed (58, 253). It is not clear that these effects are direct, and studies of the molecular structure and regulation of the cytosolic form of PLA2 have only recently begun (254). Other possible targets for G protein action are being investigated. Phosphatidylinositide 3-kinase (PI 3-kinase), for example, is an enzyme involved in plasma membrane phospholipid signaling. PI 3-kinase is known to play a role in the tyrosine kinase pathways of various oncogenes and growth factors, including platelet-derived growth factor (PDGF) and insulin, and its lipid products may have important effects on cell growth and actin polymerization (257). It is possible that the activity of PI 3-kinase is also modulated by 7-TMS receptors acting through G proteins. The increase in PI 3-kinase activity caused by activation of the formyl peptide receptor in human neutrophils is PTX-sensitive (258), while stimulation due to platelet thrombin receptors is largely PTXinsensitive (259, 260). PTX-sensitive G proteins are involved in the rapid modulation of actin polymerization and motility in human neutrophils (261), and in melanoma cell motility (222, 223, 262). Traditional second messengers do not appear to mediate these effects, but direct interaction of G proteins with the cytoskeleton, or mediation via activation of phosphatidylinositide 4-kinase have been suggested (261). The mechanism by which the Gi2-like eta protein of Drosophila rapidly alters cell morphology during gastrulation is also unresolved (101). The ability of several 7-TMS receptor agonists to stimulate proliferation or transformation in fibroblasts has been attributed to activation of G proteins (144, 263268). In the case of PTX-sensitive mitogenic effects there is strong evidence for the involvement of al2 (46, 189, 269, 270), but possible roles for other aj-subunits (271) also need to be studied. One of the recently discovered G proteins may play a role in PTX-insensitive cell growth. Preliminary results indicate that expression of mutationally activated aq in fibroblasts is transforming, but overexpression may cause cell death (43, 272). Initial attempts to relate increased DNA synthesis in fibroblasts to activation of any one of the traditional signaling pathways, including inhibition of adenylyl cyclase, stimulation of PLC, or modulation of PLA2 activity, have been unsuccessful, and it seems likely that other, unknown effector molecules are involved in the response (265, 268, 270). Tyrosine phosphorylation is known to play a role in mitogenesis induced by growth factors such as PDGF, and increased tyrosine phosphorylation of

Vol. 13, No. 3

specific cellular proteins, including mitogen-activated protein kinase (MAP kinase), has recently been proposed as a key event in the mitogenic pathway triggered by G protein-coupled receptors as well (270, 273-277). It is plausible that G proteins directly stimulate a kinase, influence an activator of MAP kinase, or inhibit a tyrosine phosphatase (270, 273, 278), but there is not yet evidence for such direct interactions. Just as our conception of the types of receptors that can activate G proteins is expanding, it is likely that we have only identified a subset of the cellular proteins that are modulated by activated G protein a- or /fy-subunits, and the search for potential effector molecules should not necessarily be restricted to the plasma membrane (119-122,125-127).

IX. G Protein Alterations in Disease States G protein function may be altered in disease states as • a result of both adaptive and maladaptive mechanisms. These include mutations in the genes encoding G protein subunits, changes in expression of G protein subunit mRNAs or proteins, posttranslational modifications of G proteins, or other undefined mechanisms (Table 3). A. Posttranslational modifications of G proteins by bacterial toxins CTX markedly reduces the intrinsic GTPase activity of the a-subunit, resulting in a constitutively active form of the protein (3). As would be predicted, this alteration raises intracellular cAMP levels independent of the normal extracellular signal (Figs. 1 and 3). The severe watery diarrhea characteristic of clinical infection with Vibrio cholerae is secondary to the direct effect of CTX on G8 in intestinal epithelial cells. It was shown more than a decade ago that rats exposed to Bordetella pertussis had an enhanced release of insulin from pancreatic 0-cells in response to secretagogues and diminished inhibition of insulin secretion by adrenergic agents (279). A specific component, named islet activating protein, which reversed a-adrenergic inhibition of adenylyl cyclase and insulin release from /3-cells, was later isolated from B. pertussis culture medium (280).. This agent, now referred to as PTX, covalently modifies several a-subunits {at, a0, an, ai2, and a^) by ADPribosylation of a cysteine residue four amino acids from the carboxy terminus (3). In contrast to the modification produced by CTX, this alteration results in the uncoupling of the modified G protein from its activating receptors) and therefore disruption of signal transduction through these G proteins. This is presumed to be the mechanism by which several clinical manifestations of pertussis (i.e. hypoglycemia and histamine sensitivity)


August,

1992


549

TABLE 3. G protein abnormalities in human disease Posttranslational Modification by Bacterial Exotoxins ADP-ribosylation of Arg201 of a8 resulting in constitutive activation in intestinal Vibrio cholerae (CTX) epithelial cells ADP-ribosylation of cysteine in carboxy-terminal region of an,2,3 and a0 resulting in Bordetella pertussis (PTX) the uncoupling of these G proteins from their receptors Diseases Associated with G Protein Gene Mutations Deficiency of functional G8, as mRNA and protein; multiple heterozygous mutations of Albright hereditary osteodystrophy a8 gene predicted to disrupt gene expression (AHO) Somatic mutations of a8 gene encoding constitutively activated

G proteins in signal transduction.

Role of G proteins in signal transduction.

Diversity of G proteins in signal transduction.

Girdin and G proteins.

Serelaxin-mediated signal transduction in human vascular cells: bell-shaped concentration-response curves reflect differential coupling to G proteins.

Receptor-effector coupling by G proteins.

Signal transduction in Coprinus congregatus: evidence for the involvement of G proteins in blue light photomorphogenesis.

SIGNAL TRANSDUCTION. Structural basis for nucleotide exchange in heterotrimeric G proteins.

G proteins: implications for psychiatry.

Autophagy signal transduction by ATG proteins: from hierarchies to networks.

Transduction of plant signal molecules by the Rhizobium NodD proteins.

Mitogenic signal transduction in normal and transformed 32D hematopoietic cells.

Single-cell analysis of G-protein signal transduction.

Signal transduction by guanylyl cyclases.

GTP binding proteins and signal transduction in the human neutrophil.

PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation.

PI3K-C2α: One enzyme for two products coupling vesicle trafficking and signal transduction.

Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum.

N-Terminal Signal Peptides of G Protein-Coupled Receptors: Significance for Receptor Biosynthesis, Trafficking, and Signal Transduction.

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases.

The role of ras proteins in insulin signal transduction.

Post-translational modification of P II signal transduction proteins.

GTP-binding proteins are restricted to signal transduction sites.